Wilms' tumor 1-associating protein (WTAP) plays an important role in physiological processes and the development of tumor such as cell cycle regulation. The regulation of cell cycle is mainly dependent on cyclins and cyclin-dependent protein kinases (CDKs). Recent studies have shown that CDKs are closely related to the tumor diagnosis, progression and response to treatment. However, their specific biological roles and related mechanism in renal cell carcinoma (RCC) remain unknown.

Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake via degradation of TXNIP is largely dependent upon adipose triglyceride lipase (ATGL) and not hormone-sensitive lipase or monoacylglycerol lipase. Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake.

CDK5RAP3 (CDK5 regulatory subunit associated protein 3) was originally identified as a binding protein of CDK5. It is a crucial gene controlling biological functions, such as cell proliferation, apoptosis, invasion, and metastasis. Although previous studies have also shown that CDK5RAP3 is involved in a variety of signalling pathways, however, the mechanism of CDK5RAP3 remains largely undefined. This study utilized MEFs from conditional knockout mice to inhibit CDK5RAP3 and knockdown CDK5RAP3 in MCF7 to explore the role of CDK5RAP3 in cell growth, mitosis, and cell death.

Clade 2.3.4.4 avian H5Ny viruses, namely H5N2, H5N6, and H5N8, have exhibited unprecedented intercontinental spread in poultry. Among them, only H5N6 viruses are frequently reported to infect mammals and cause serious human infections. In this study, the genetic and biological characteristics of surface hemagglutinin (HA) from clade 2.3.4.4 H5Ny avian influenza viruses (AIVs) were examined for adaptation in mammalian infection. Phylogenetic analysis identified an amino acid (AA) deletion at position 131 of HA as a distinctive feature of H5N6 virus isolated from human patients. This single AA deletion was found to enhance H5N6 virus replication and pathogenicity in vitro and in mammalian hosts (mice and ferrets) through HA protein acid and thermal stabilization that resulted in reduced pH threshold from pH 5.7 to 5.5 for viral-endosomal membrane fusion. Mass spectrometry and crystal structure revealed that the AA deletion in HA at position 131 introduced an N-linked glycosylation site at 129, which increases compactness between HA monomers, thus stabilizes the trimeric structure. Our findings provide a molecular understanding of how HA protein stabilization promotes cross-species avian H5N6 virus infection to mammalian hosts.

Ribosomal S6 Kinase 2 (RSK2) is a member of the p90(RSK) family of serine/threonine kinases, which are widely expressed and respond to many growth factors, peptide hormones, and neurotransmitters. Loss-of function mutations in the RPS6KA3 gene, which encodes the RSK2 protein, have been implicated in Coffin-Lowry Syndrome (CLS), an X-linked mental retardation disorder associated with cognitive deficits and behavioral impairments. However, the cellular and molecular mechanisms underlying this neurological disorder are not known. Recent evidence suggests that defective DNA damage signaling might be associated with neurological disorders, but the role of RSK2 in the DNA damage pathway remains to be elucidated. Here, we show that Adriamycin-induced DNA damage leads to the phosphorylation of RSK2 at Ser227 and Thr577 in the chromatin fraction, promotes RSK2 nuclear translocation, and enhances RSK2 and Atm interactions in the nuclear fraction. Furthermore, using RSK2 knockout mouse fibroblasts and RSK2-deficient cells from CLS patients, we demonstrate that ablation of RSK2 impairs the phosphorylation of Atm at Ser1981 and the phosphorylation of p53 at Ser18 (mouse) or Ser15 (human) in response to genotoxic stress. We also show that RSK2 affects p53-mediated downstream cellular events in response to DNA damage, that RSK2 knockout relieves cell cycle arrest at the G2/M phase, and that an increased number of γH2AX foci, which are associated with defects in DNA repair, are present in RSK2-deficient cells. Taken together, our findings demonstrated that RSK2 plays an important role in the DNA damage pathway that maintains genomic stability by mediating cell cycle progression and DNA repair.

The widely used SiO2 and TiO2 nanoparticles (NPs) can accumulate in industrial wastewaters, thereby posing challenge to biological wastewater treatment processes. In this work, the performance of aerobic granular sludge (AGS) reactors fed with wastewater containing 50 mg L-1 SiO2 and TiO2 NPs were investigated. The results showed that the granules could resist the NPs in wastewater (no disintegration of granules was observed). SiO2 NPs had a negative effect on the settleability of granules, with the SVI30 increased by 64.5% and protein secretion decreased by 29.9%. To the contrary, the settleability of granules was improved in the presence of TiO2 NPs due to the increase of the protein secretion. Possibly because of the compact and layered structure of granules, in the presence of both types of NPs, no obvious reduction of the overall removal efficiency of organics was found, and nanoparticle-resistant strains were enriched. The overall nitrification and denitrification efficiencies were hardly affected by SiO2 NPs while significantly inhibited by TiO2 NPs. Some functional genera, such as Hyphomicrobium and Acidovorax, showed growth inhibition with TiO2 NPs, which might be responsible for the reduction of nitrification and denitrification efficiencies.

Inhibitors of the CDK family of proteins have been approved for the treatment of a variety of tumours; however, the development of new drugs administered in combination with CDK inhibitors is expected to improve the therapeutic effect. We identified the function of suppressor of cytokine signalling 1 (SOCS1) in hepatocellular carcinoma (HCC) cell models and the xenograft mouse model. When SOCS1 expression was artificially upregulated, HCC cell lines were arrested at the G1-S transition in the cell cycle. Interestingly, during this process, total CyclinD1 protein increased, but the effective proportion decreased. We found that the deficiency of CyclinD1 in the nucleus is probably due to the decrease in the stability of nuclear CyclinD1 caused by the ubiquitin-based degradation of P21, thus inhibiting the progression of the cell cycle to S phase. After P21 expression was increased, the levels of the component that inactivates CyclinD1 decreased as expected. It showed that P21 has a partial promoting effect on cancer. SOCS1 is a good indicator of prognosis, tumour size and long-term survival after resection. SOCS1 is expected to become a drug target in combined with CDK family inhibitors.

We analyzed the effects of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-12 on the degree of carotid atherosclerosis (CAS) and plaque stability, and investigated their correlations with cardiovascular and cerebrovascular events (CCEs). Two hundred CAS patients were enrolled. Carotid intima-media thickness (IMT) was measured using ultrasonic examination. Patients were divided into the no plaque group (NP group), stable plaque group (SP group), and vulnerable plaque group (VP group). The Crouse method was used for the evaluation of plaque scores. Additionally, 60 healthy subjects were enrolled as the control group. Serum triacylglycerol (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) were analyzed. The serum protein levels of MMP-1, MMP-3, and MMP-12 were measured by western blotting. The frequency of CCEs within 2 years was recorded, and its correlation with MMP-1, MMP-3, and MMP-12 was analyzed. The CAS plaque scores in the SP and VP groups were significantly increased compared with the NP group, and the difference between the SP and VP groups was significant. The levels of TC, TG, LDL-C, and HDL-C of CAS patients were significantly increased compared with those in the control group, but the differences in these indexes between the patient groups were not significant. Western blotting showed that the levels of MMP-1, MMP-3, and MMP-12 in the patient groups were significantly increased compared with those in the control group, and the protein levels in the VP group were significantly higher than those in the SP and NP groups. Additionally, the levels of MMP-1, MMP-3, and MMP-12 had significantly positive correlations with the occurrence of CCEs in CAS patients. In conclusion, MMP-1, MMP-3, and MMP-12 are positively correlated with CCEs in CAS patients. They can be used as markers for the clinical diagnosis and prognosis of CAS.

Doxorubicin (DOX) is commonly used for chemotherapy; however, its clinical value is extremely dampened because of the fatal cardiotoxicity. Leucine zipper protein 1 (LUZP1) plays critical roles in cardiovascular development, and this study is designed for determining its function and mechanism in DOX-induced cardiotoxicity.

Poly(ethylene terephthalate) (PET) is used widely by human beings, but is very difficult to degrade. Up to now, the PET degradation effect of PETase from Ideonella sakaiensis 201-F6 (IsPETase) variants with low stability and activity was not ideal. In this study, a mutation design tool, Premuse, was developed to integrate the sequence alignment and quantitative selection of the preferred mutations based on natural sequence evolution. Ten single point mutants were selected from 1486 homologous sequences using Premuse, and then two mutations (W159H and F229Y) with improved stability were screened from them. The derived double point mutant, W159H/F229Y, exhibited a strikingly enhanced enzymatic performance. Its Tm and catalytic efficiency values (kcat/Km) respectively increased by 10.4 °C and 2.0-fold using p-NPP as the substrate compared with wild type. The degradation activity for amorphous PET was increased by almost 40-fold in comparison with wild type at 40 °C in 24 h. Additionally, the variant could catalyze biodegradation of PET bottle preform at a mean rate of 23.4 mgPET/h/mgenzyme. This study allowed us to design the mutation more efficiently, and provides a tool for achieving biodegradation of PET pollution under mild natural environments.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

Aging of the organism is associated diminished response to external stimuli including weakened immune function, resulting in diseases that impair health and lifespan. Several dietary restriction modalities have been reported to improve health and lifespan in different animal models, but it is unknown whether any of the lifespan-extending dietary treatments could be combined to achieve an additive effect. Here, we investigated the effects of halving amino acids components in the HUNTaa diet, a synthetic medium known to extend lifespan in Drosophila. We found that dietary restriction by halving the entire amino acid components (DR group) could further extend lifespan and improve resistance to oxidative stress, desiccation stress, and starvation than flies on HUNTaa diet alone (wt group). Transcriptome analysis of Drosophila at 40, 60, and 80 days of age revealed that genes related to cell proliferation and metabolism decreased with age in the wt group, whereas background stimulus response and amino acid metabolism increased with age. However, these trends differed in the DR group, that is, the DR flies had downregulated stress response genes, including reduced background immune activation. Infection experiments demonstrated that these flies survived longer after feeding infection with Serratia marcescens and Enterococcus faecalis, suggesting that these flies had stronger immune function, and therefore reduced immune senescence. These results demonstrated that halving the entire amino acid components in the HUNTaa diet further extended health and lifespan and suggested that lifespan-extending diet and dietary restriction treatment could be combined to achieve additive beneficial results.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2-3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC(50) of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood-brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cells, potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73) were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel therapeutic candidates for ALS.

It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro, and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo, purified rBanLec-1 exhibited more significant effects on TMV infection in vitro. Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.

An efficient supply of reducing equivalent is essential for chemicals production by engineered microbes. In phototrophic microbes, the NADPH generated from photosynthesis is the dominant form of reducing equivalent. However, most dehydrogenases prefer to utilize NADH as a cofactor. Thus, sufficient NADH supply is crucial to produce dehydrogenase-derived chemicals in cyanobacteria. Photosynthetic electron is the sole energy source and excess electrons are wasted in the light reactions of photosynthesis.

Hepatocellular carcinoma (HCC) is often diagnosed in patients with advanced disease who are ineligible for curative surgical therapies. Sorafenib is a first-line agent approved for the treatment of advanced HCC. However, the frequent resistance of HCC cells to sorafenib greatly reduces its efficacy. Herein, we describe a novel long non-coding RNA (lncRNA) conferring sorafenib resistance. Long intergenic non-protein coding RNA 1273 (LINC01273) was significantly overexpressed in human HCC and sorafenib-resistant tissues, linking it to poor overall and relapse-free survival. We established sorafenib-resistant Huh7 (Huh7-SR) and SMMC-7721 (SMMC-7721-SR) cells, and found that the knockdown of LINC01273 repressed the viability, colony formation, and DNA synthesis rate of Huh7-SR and SMMC-7721-SR cells. The level of N6-methyladenosine (m6A) in sorafenib-resistant HCC cells was significantly decreased, which was rescued by LINC01273 silencing. Mechanistically, LINC01273 complementarity bound to miR-600, served as a 'reservoir' increasing miR-600 stability, and facilitating miR-600 targeting methyltransferase 3 (METTL3), a m6A 'writer', resulting in reducing METTL3 level. In addition, LINC01273 was modified with m6A, METTL3 increased LINC01273 m6A modification, followed by LINC01273 decay in the presence of YTHDF2, a m6A 'reader'. Our findings reveal the key role of LINC01273 in sorafenib-resistant HCC cells, and targeting of the newly identified LINC01273/miR-600/METTL3 feedback regulatory axis may be a promising effective intervention for HCC patients with sorafenib resistance.

Decreased intercellular adhesion is a key step in the metastasis and recurrence of many cancers, including hepatocellular carcinoma (HCC). SVEP1 is an important cell adhesion molecule that plays a key role in regulating intercellular adhesion and embryonic lymphatic development. However, the expression patterns and roles of SVEP1 in HCC are still largely unknown. We identified SVEP1 expression by analyzing 220 HCC samples from our cancer center. TCGA and GEO online-databases were used for data calibration and validation. SVEP1 was differentially expressed in two groups of HCCs with different risks of recurrence and was deemed as an independent risk factor for the prognosis of HCC. The expression of SVEP1 is negatively related to the proliferation and metastasis of HCC. Downregulation of SVEP1 expression promoted in vitro HCC cell migration, chemotaxis, invasion and proliferation, as well as in vivo tumor growth, local invasion and metastasis in a mouse model. Bioinformatic analysis and RT-PCR results showed that miR-1269b expression is negatively correlated with the SVEP1 expression and the prognosis of HCC patients. Further experiments showed that miR-1269b directly targets and downregulates the expression of SVEP1, which further induces the phosphorylation of Akt at thr308. These regulatory effects ultimately mediate the proliferation and metastasis of HCC cells. SVEP1 could serve as a promising prognostic marker of HCC. MiR-1269b downregulates SVEP1 expression and promotes HCC proliferation and metastasis likely through the PI3k/Akt signaling pathway.

ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

Sugar transporter protein (STP) genes are involved in multiple biological processes, such as plant responses to various stresses. However, systematic analysis and functional information of STP family genes in Brassica oleracea are very limited. A comprehensive analysis was carried out to identify BoSTP genes and dissect their phylogenetic relationships and to investigate the expression profiles in different organs and in response to the clubroot disease. A total of 22 BoSTP genes were identified in the B. oleracea genome and they were further classified into four clades based on the phylogenetic analysis. All the BoSTP proteins harbored the conserved sugar transporter (Sugar_tr, PF00083) domain, and the majority of them contained 12 transmembrane helices (TMHs). Rates of synonymous substitution in B. oleracea relative to Arabidopsis thaliana indicated that STP genes of B. oleracea diverged from those of A. thaliana approximately 16.3 million years ago. Expression profiles of the BoSTP genes in different organs derived from RNA-Seq data indicated that a large number of the BoSTP genes were expressed in specific organs. Additionally, the expression of BoSTP4b and BoSTP12 genes were induced in roots of the clubroot-susceptible cabbage (CS-JF1) at 28 days after inoculation with Plasmodiophora brassicae, compared with mock-inoculated plants. We speculated that the two BoSTPs might be involved in monosaccharide unloading and carbon partitioning associated with P. brassicae colonization in CS-JF1. Subcellular localization analysis indicated that the two BoSTP proteins were localized in the cell membrane. This study provides insights into the evolution and potential functions of BoSTPs.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.